Characterization of West Nile virus Koutango lineage from phlebotomine sandflies in Kenya

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. This study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12th -16th March 2021 and 16th -20th March 2023 from six villages each in Baringo and Isiolo Counties, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus and site of collection. Virus isolation was performed in Vero cells. Viral genomes were determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the virus's evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian Vero E6 and mosquito C6/36 cells. We report the isolation of WN-KOUTV from Salabani in Baringo and Aremet in Isiolo, Kenya. The isolated WN-KOUTVs clustered with previously identified WN-KOUTV strains. Comparative analysis revealed a unique amino acid at NS5 653. The WN-KOUTV lineage as a whole is under purifying selective pressure, with diversifying pressure acting at site NS3 267. The current WN-KOUTV replicated in Vero E6 and C6/36 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. Subsequent isolations of WN-KOUTV in phlebotomine sandflies suggest potential vectors, however, vector competence studies would confirm this. Replication in mammalian and insect cell lines suggests there may exist a vector/host relationship. We speculate the close genetic relationship of WN-KOUTV strains from East and West Africa may potentially be enabled by bird migratory routes between the two regions. If proven, this could point to a potential future pandemic pathway for this virus.